Comparative Extracellular Proteomics of Reveals Iron-Regulated Secreted Proteins as Potential Vaccine Candidates.

In our previous study, several iron-related outer membrane proteins in , a serious pathogen of farmed fish, conferred high immunoprotectivity to fish, and were proposed as potential vaccine candidates. However, the protective efficacy of these extracellular proteins against remains largely unknown. Here, we identified secreted proteins that were differentially expressed in LP-2 in response to iron starvation using an iTRAQ-based quantitative proteomics method. We identified 341 proteins, of which 9 were upregulated in response to iron starvation and 24 were downregulated. Many of the differently expressed proteins were associated with protease activity. We confirmed our proteomics results with Western blotting and qPCR. We constructed three mutants by knocking out three genes encoding differentially expressed proteins (Δ, Δ, and Δ). The physiological characteristics of these mutants were investigated. In all these mutant strains, protease activity decreased, and Δ, and Δ were less virulent in zebrafish. This indicated that the proteins encoded by these genes may play important roles in bacterial infection. We next evaluated the immune response provoked by the six iron-related recombinant proteins (ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641) in zebrafish as well as the immunization efficacy of these proteins. Immunization with these proteins significantly increased the zebrafish immune response. In addition, the relative percent survival (RPS) of the immunized zebrafish was 50-80% when challenged with three virulent strains, respectively. Thus, these extracellular secreted proteins might be effective vaccine candidates against infection in fish.